This invention relates generally to novel compositions comprising encapsulated, fluorescent nanocrystals. More particularly, the present invention relates to the use of a vesicle or capsid to encapsulate fluorescent nanocrystals in forming water-soluble fluorescent nanocrystals.
Nonisotopic detection systems have become a preferred mode in scientific research and clinical diagnostics for the detection of biomolecules using various assays including, but not limited to, flow cytometry, nucleic acid hybridization, DNA sequencing, nucleic acid amplification, microarrays, immunoassays, histochemistry, and functional assays involving living cells. In particular, while fluorescent organic molecules such as fluorescein and phycoerythrin are used frequently in detection systems, there are disadvantages in using these molecules in combination. For example, each type of fluorescent molecule typically requires excitation with photons of a different wavelength as compared to that required for another type of fluorescent molecule. However, even when a single light source is used to provide a single excitation wavelength (in view of the spectral line width), often there is insufficient spectral spacing between the emission optima of different fluorescent molecules to permit individual and quantitative detection without substantial spectral overlap. Additionally, conventional fluorescent molecules have limited fluorescence intensity. Further, currently available nonisotopic detection systems typically are limited in sensitivity due to the finite number of nonisotopic molecules which can be used to label a biomolecule to be detected.
Doped metal oxide (xe2x80x9cdMOxe2x80x9d) nanocrystals are nanocrystals that can be excited with a single excitation light source resulting in a detectable fluorescence emission of high quantum yield (e.g., a single quantum dot having at a fluorescence intensity that may be a log or more greater than that a molecule of a conventional fluorescent dye) and with a discrete fluorescence peak. Typically, they have a substantially uniform size of less than 200 Angstroms, and preferably have a substantially uniform size in the range of sizes of from about 1 nm to about 5 nm, or less than 1 nm. In that regard, dMO nanocrystals are preferably comprised of metal oxides doped with one or more rare earth elements, wherein the dopant comprising the rare earth element is capable of being excited (e.g., with ultraviolet light) to produce a narrow spectrum of fluorescence emission (typically more narrow than the spectrum of fluorescence emission emitted by a semiconductor nanocrystal). Such dMO nanocrystals are well known in the art. However, a desirable feature of dMO nanocrystals when used for nonisotopic detection applications is that the nanocrystals be made water-soluble. xe2x80x9cWater-solublexe2x80x9d is used herein to mean that the nanocrystals are sufficiently soluble or suspendable in an aqueous-based solution including, but not limited to, water, water-based solutions, and buffer solutions, that are used in detection processes, as known by those skilled in the diagnostic art.
Semiconductor nanocrystals are quantum dots that can be excited with a single excitation light source resulting in a detectable fluorescence emission of high quantum yield (e.g., a single quantum dot having at a fluorescence intensity that may be a log or more greater than that a molecule of a conventional fluorescent dye) and with a discrete fluorescence peak. Typically, they have a substantially uniform size of less than 200 Angstroms, and preferably have a substantially uniform size in the range of sizes of from about 1 nm to about 5 nm, or less than 1 nm. In that regard, quantum dots are preferably comprised of a Group II-VI semiconductor material (of which ZnS, and CdSe are illustrative examples), or a Group III-V semiconductor material (of which GaAs is an illustrative example). Such quantum dots are well known in the art. However, a desirable feature of quantum dots when used for nonisotopic detection applications is that the quantum dots be made water-soluble. Current methods of making semiconductor nanocrystals water-soluble is to add to the semiconductor nanocrystal a layer comprising mercaptocarboxylic acid (Chen and Nie, 1998, Science 281:2016-2018), or silica (U.S. Pat. No. 5,990,479), or one or more layers of amino acids (U.S. Pat. No. 6,114,038). Depending on which layer composition is used, the treated nanocrystal may have limited stability in an aqueous solution, particularly when exposed to air (oxygen) and/or light. More particularly, oxygen and light can cause the molecules comprising the layer to become oxidized, thereby forming disulfides which destabilize the attachment of the layer molecules to the semiconductor nanocrystals. Thus, oxidation may cause the layer molecules to become detached from the surface of the quantum dots, thereby exposing the surface of the quantum dots which may result in xe2x80x9cdestabilized quantum dotsxe2x80x9d. Destabilized quantum dots form aggregates when they interact together, and the formation of such aggregates eventually leads to irreversible flocculation of the quantum dots. Additionally, depending on the layer composition, it can cause non-specific binding, particularly to one or more molecules in a sample other than the target molecule, which is not desirable in a detection assay.
Hence, there is a need to provide alternative forms of water-soluble, fluorescent nanocrystals.
It is a primary object of the present invention to provide fluorescent nanocrystals which are encapsulated by a vesicle or capsid comprising a liposome.
It is another object of the present invention to provide fluorescent nanocrystals which are encapsulated by or trapped within a vesicle or capsid comprising a liposome, and wherein the surface of the liposome is functionalized with surface groups comprising a reactive functionality that may be used to form a bond with one or more molecules of an affinity molecule which has a reactive functionality which is capable of forming a bond with the surface groups of the liposome.
It is another object of the present invention to provide a fluorescent nanocrystal which comprises one or more fluorescent nanocrystals encapsulated by or trapped within a liposome, and wherein the surface of the liposome is functionalized with surface groups comprising one or more reactive functionalities.
It is another object of the present invention to provide a functionalized, encapsulated fluorescent nanocrystal which comprises one or more fluorescent nanocrystals encapsulated by or trapped within a liposome which is functionalized by the addition of one or more affinity molecules.
It is further object of the present invention to provide a functionalized, encapsulated fluorescent nanocrystal which comprises one or more fluorescent nanocrystals encapsulated by or trapped within a liposome, and wherein the liposome portion may be disrupted to release the fluorescent nanocrystals in a method of xe2x80x9cquenchingxe2x80x9d the fluorescence in a reaction.
Definitions
By the term xe2x80x9cfluorescent nanocrystalsxe2x80x9d is meant, for purposes of the specification and claims to refer to fluorescent nanocrystals comprised of doped metal oxide nanocrystals, semiconductor nanocrystals, or a combination thereof.
By the terms xe2x80x9cdoped metal oxide nanocrystalsxe2x80x9d or xe2x80x9cdMO nanocrystalsxe2x80x9d is meant, for purposes of the specification and claims to refer to nanocrystals comprised of: a metal oxide, and a dopant comprised of one or more rare earth elements. For example, suitable metal oxides include, but are not limited to, yttrium oxide (Y2O3), zirconium oxide (ZrO2), zinc oxide (ZnO), copper oxide (CuO or Cu2O), gadolinium oxide (Gd2O3), praseodymium oxide (Pr2O3), lanthanum oxide (La2O3), and alloys thereof. The rare earth element comprises an element selected from the Lanthanide series and includes, but is not limited to, europium (Eu), cerium (Ce), neodymium (Nd), samarium (Sm), terbium (Tb), gadolinium (Gd), holmium (Ho), thulium (Tm), an oxide thereof, and a combination thereof. As known to those skilled in the art, depending on the dopant, an energized dMO nanocrystal is capable of emitting light of a particular color. Thus, the nature of the rare earth or rare earths are selected in consequence to the color sought to be imparted (emitted) by a functionalized, encapsulated dMO nanocrystal according to the present invention. A given rare earth or rare earth combination has a given color, thereby permitting the provision of functionalized, encapsulated dMO nanocrystals, each of which may emit (with a narrow emission peak) a color over an entire range of colors by adjusting the nature of the dopant, the concentration of the dopant, or a combination thereof. For example, the emission color and brightness (e.g., intensity) of a dMO nanocrystal comprising Y2O3:Eu may depend on the concentration of Eu; e.g., emission color may shift from yellow to red with increasing Eu concentration. For purposes of illustration only, representative colors which may be provided are listed in Table 1.
Methods for making dMO nanocrystals are known to include, but are not limited to a sol-gel process (see, e.g., U.S. Pat. No. 5,637,258), and an organometallic reaction. As will be apparent to one skilled in the art, the dopant (e.g., one or more rare earth elements) are incorporated into the dMO nanocrystal in a sufficient amount to permit the dMO nanocrystal to be put to practical use in fluorescence detection as described herein in more detail. An insufficient amount comprises either too little dopant which would fail to emit sufficient detectable fluorescence, or too much dopant which would cause reduced fluorescence due to concentration quenching. In a preferred embodiment, the amount of dopant in a dMO nanocrystal is a molar amount in the dMO nanocrystal selected in the range of from about 0.1% to about 25%.
By the term xe2x80x9csemiconductor nanocrystalsxe2x80x9d is meant, for purposes of the specification and claims to refer to quantum dots (crystalline semiconductors) comprised of a core comprised of at least one of a Group II-VI semiconductor material (of which ZnS, and CdSe are illustrative examples), or a Group III-V semiconductor material (of which GaAs is an illustrative example), a Group IV semiconductor material, or a combination thereof. In a preferred embodiment, the core of the quantum dots may be passivated with an semiconductor overlayering (xe2x80x9cshellxe2x80x9d) uniformly deposited thereon. For example, a Group II-VI semiconductor core may be passivated with a Group II-VI semiconductor shell (e.g., a ZnS or CdSe core may be passivated with a shell comprised of YZ wherein Y is Cd or Zn, and Z is S, or Se). As known to those skilled in the art, the size of the semiconductor core correlates with the spectral range of emission, as illustrated in Table 1 for CdSe.
Methods for making semiconductor nanocrystals are known in the art. A preferred method of making semiconductor nanocrystals is by a continuous flow process (U.S. Pat. No. 6,179,912, the disclosure of which is herein incorporated by reference).
By the term xe2x80x9caffinity moleculexe2x80x9d is meant, for purposes of the specification and claims, to mean a molecule which is capable of binding to another molecule; and in a preferred embodiment, has binding specificity and avidity for a target molecule. In general, affinity molecules are known to those skilled in the art to include, but are not limited to, lectins or fragments (or derivatives) thereof which retain binding function; monoclonal antibodies (xe2x80x9cmAbxe2x80x9d, including chimeric or genetically modified monoclonal antibodies (e.g., xe2x80x9chumanizedxe2x80x9d)); peptides; aptamers; nucleobases (synthetic, natural, or modified); nucleic acid molecules (including, but not limited to, single stranded RNA or single-stranded DNA, or single-stranded nucleic acid hybrids); avidin, or streptavidin, or avidin derivatives; and the like. The invention may be practiced using a preferred affinity molecule to the exclusion of affinity molecules other than the preferred affinity molecule. The term xe2x80x9cmonoclonal antibodyxe2x80x9d is also used herein, for purposes of the specification and claims, to include immunoreactive fragments or derivatives derived from a mAb molecule, which fragments or derivatives retain all or a portion of the binding function of the whole mAb molecule. Such immunoreactive fragments or derivatives are known to those skilled in the art to include F(abxe2x80x2)2, Fabxe2x80x2, Fab, Fv, scFV, Fdxe2x80x2 and Fd fragments. Methods for producing the various fragments or derivatives from mAbs are well known in the art. The construction of chimeric antibodies is now a straightforward procedure in which the chimeric antibody is made by joining the murine variable region to a human constant region. Additionally, xe2x80x9chumanizedxe2x80x9d antibodies may be made by joining the hypervariable regions of the murine monoclonal antibody to a constant region and portions of variable region (light chain and heavy chain) sequences of human immunoglobulins using one of several techniques known in the art. Aptamers can be made using methods described in U.S. Pat. No. 5,789,157 (herein incorporated by reference). Lectins, and fragments thereof, are commercially available. Lectins are known to those skilled in the art, and are commercially available.
By the term xe2x80x9cnucleobasexe2x80x9d is meant, for purposes of the specification and claims to refer to a nucleic acid moiety including, but not limited to: nucleosides (including derivatives, or functional equivalents thereof, and synthetic or modified nucleosides, and particularly, a nucleoside comprising a reactive functionality (e.g., free amino group or carboxyl group); nucleotides (including dNTPs, ddNTPs, derivatives or functional equivalents thereof, and particularly, a nucleotide comprising a reactive functionality (e.g., free amino group or carboxyl group); acyclonucleoside triphosphates (see, e.g., U.S. Pat. No. 5,558,991); 3xe2x80x2 (2xe2x80x2)-amino-modified nucleosides, 3xe2x80x2 (2xe2x80x2)-amino-modified nucleotides, 3xe2x80x2 (2xe2x80x2)-thiol-modified nucleosides, 3xe2x80x2 (2xe2x80x2)-thiol-modified nucleotides (see, e.g., U.S. Pat. No. 5,679,785); alkynylamino-nucleotides (see, e.g., as a chain terminator, U.S. Pat. No. 5,151,507); and nucleoside thiotriphosphates (see, e.g., U.S. Pat. No. 5,187,085).
By the term xe2x80x9creactive functionalityxe2x80x9d is meant, for purposes of the specification and claims, to refer to a free chemical group which can bond or associate with a chemical-reactive group (reactive with the free chemical groups). In a preferred embodiment, the resultant bond or association is of sufficient stability to withstand conditions encountered in a method of detection, as known in the art. Free chemical groups include, but are not limited to a thiol, carboxyl, hydroxyl, amino, amine, sulfo, phosphate, or the like; whereas chemical-reactive groups include, but are not limited to, thiol-reactive group, carboxyl-reactive group, hydroxyl-reactive group, amino-reactive group, amine-reactive group, sulfo-reactive group, or the like.
By the term xe2x80x9cliposomexe2x80x9d is meant, for purposes of the specification and claims, to refer to a generally spherical vesicle or capsid generally comprised of amphipathic molecules (e.g., having both a hydrophobic (nonpolar) portion and a hydrophilic (polar) portion). Typically, the liposome can be produced as a single (unilamellar) closed bilayer or a multicompartment (multilamellar) closed bilayer. The liposome can be formed by natural lipids, synthetic lipids, or a combination thereof. In a preferred embodiment, the liposome comprises one or more phospholipids. In a more preferred embodiment, the liposome is substituted with one or more conventional additives (xe2x80x9ca component for substitutionxe2x80x9d), wherein the one or more additives are selected from the group consisting of a membrane stabilizer, an isotonic agent (e.g., sugars, sodium chloride, polyalcohols such as mannitol, sorbitol, and the like), a pH adjusting agent (e.g., a base, a basic amino acid, an acidic amino acid, sodium phosphate, sodium carbonate, and the like, present in an amount to adjust the liposome to a desired pH), an aggregation minimizer (e.g., a surfactant (e.g., polysorbates, poloxamers), polysaccharide, liposomal surface carboxyl groups, and the like), an affinity molecule, an amino acid, and a combination thereof. As apparent to one skilled in the art, and depending on the lipid composition and the composition of the component for substitution, the one or more components for substitution may be added during the formation of the liposome, may be added after the formation of the liposome, or a combination thereof. A preferred component for substitution of the liposome may be used to the exclusion of components other than the preferred component. For example, a membrane stabilizer is added in an effective amount to increase the stability of a liposome. Stability refers to one or more of membrane integrity, ability to withstand heat (e.g., a temperature above room temperature, and preferably a temperature in the range of from about 35xc2x0 C. to about 100xc2x0 C.), ability to withstand oxygen (e.g., as exposed during normal use conditions), ability to withstand light (e.g., as exposed during normal use conditions), and a combination thereof. A membrane stabilizer may comprise one or more sterols (e.g., cholesterol), one or more fatty acids, one or more amino acids, and a combination thereof. Also, stability, with respect to exposure to oxidation, may be enhanced by nitrogen gas substitution using methods known on the art. Lipids known in the art for forming liposomes include, but are not limited to, lecithin (soy or egg; phosphatidylcholine), dipalmitoylphosphatidylcholine, dimyristoylphosphatidylcholine, distearoylphosphatidylcholine, dicetylphosphate, phosphatidylglycerol, hydrogenated phosphatidylcholine, phosphatidic acid, cholesterol, phosphatidylinositol, a glycolipid, phosphatidylethanolamine, phosphatidylserine, a maleimidyl-derivatized phospholipid (e.g., N-[4(p-maleimidophenyl)butyryl] phosphatidylethanolamine), dioleylphosphatidylcholine, dipalmitoylphosphatidylglycerol, dimyristoylphosphatidic acid, and a combination thereof. It will be apparent to one skilled in the art that the ratio of the one or more lipids and the one or more components for substitution will depend on factors including, but not limited to, the composition of the lipids, the intended function of each lipid (e.g., the reason for its inclusion in the liposome), the composition of the component for substitution, the intended function of each component for substitution (e.g., the reason for its inclusion in the liposome), and the desired properties of the liposome portion of a functionalized, encapsulated fluorescent nanocrystal (e.g., size of interior space or xe2x80x9ccapture volumexe2x80x9d, pH range of stability, temperature range of stability, desired surface charge, desired surface free chemical group). In that regard, as known to those skilled in the art, a particular lipid or lipid combination, when used to form a liposome, can offer particular benefits. For example, inclusion of phosphatidylglycerol in combination with other lipids (e.g., with phosphatidylcholine and cholesterol, ratio of 1:9:8) imparts a negative charge to the liposome which increases intralamellar spacing (capture volume), reduces aggregation, and facilitates initial hydration of the lipid. In a preferred embodiment, the liposomes encapsulating the fluorescent nanocrystals are stable at a neutral pH of from about 6 to about 7; and in a more preferred embodiment, are stable in a broad pH range of from about 4 to about 12. A preferred liposome (content and composition) may be formed as part of the functionalized, encapsulated fluorescent nanocrystals according to the present invention to the exclusion of liposomes other than the preferred liposome.
By the term xe2x80x9cfunctionalized, encapsulated fluorescent nanocrystalxe2x80x9d is meant, for purposes of the specification and claims to refer to one or more fluorescent nanocrystals which have been encapsulated (e.g., without establishing a chemical linkage or bond between the one or more fluorescent nanocrystals and the liposome) by a liposome; wherein the outer surface of the liposome is functionalized with surface groups comprising one or more reactive functionalities, one or more affinity molecules, or a combination thereof; and wherein the functionalized, encapsulated fluorescent nanocrystal is water-soluble. In one preferred embodiment, a single fluorescent nanocrystal is encapsulated by the liposome. In another preferred embodiment, a plurality of fluorescent nanocrystals are encapsulated by a liposome. It will be apparent to one skilled in the art that the number of fluorescent nanocrystals encapsulated per liposome can be controlled by factors that include, but are not limited to, the size of the liposome formed, the method in which the fluorescent nanocrystals are encapsulated, post production processing by size exclusion, and the ratio of fluorescent nanocrystals to lipid mixture during formation. It will also be apparent to one skilled in the art, that where a plurality of fluorescent nanocrystals are encapsulated by a liposome, the fluorescent nanocrystals may be homogeneous (i.e., capable of fluorescing essentially the same color) or may be heterogenous (e.g., comprising different populations wherein each population is capable of fluorescing a different (spectrally distinguishable) color than another population of fluorescent nanocrystal that is encapsulated).
By the term xe2x80x9cstrand synthesisxe2x80x9d is meant for purposes of the specification and claims to refer to the production of one more strands, or portions thereof, such as through enzymatic copying by an enzyme which replicates nucleic acids in a template-directed manner. There is no particular size, length or content limitations for the strand. Thus, xe2x80x9cstrand synthesisxe2x80x9d encompasses processes including, but not limited to, nucleic acid amplification, DNA sequencing, fill-in reactions, reverse transcription, in vitro mutagenesis, cycled chain termination sequence reactions, cycled primer extension reactions, random primer extension reactions, nick translations, primer elongation, methods for determining the presence and quantifying the number of di- and trinucleotide repeats (see, e.g., U.S. Pat. No. 5,650,277), and DNA typing with short tandem repeat polymorphisms (see, e.g., U.S. Pat. No. 5,364,759). The nucleic acid composition of the strand synthesized may be selected from molecules which include nucleobases; and more preferably, ribonucleotides (RNA), or deoxyribonucleotides (DNA).
This invention relates to compositions comprising functionalized, encapsulated fluorescent nanocrystals. Preferably, the functionalized, encapsulated fluorescent nanocrystals are water-soluble, and can be stored without significant leakage (of the one or more fluorescent nanocrystals from the liposome) over long periods of time. The outer surface of the liposome portion (e.g., polar head groups in contact with the surrounding aqueous environment) is functionalized with surface groups comprising one or more reactive functionalities, one or more affinity molecules, or a combination thereof. As known to those skilled in the art, dMO nanocrystals and semiconductor nanocrystals are generally soluble in organic solvents, and have limited or no solubility in aqueous environments. Thus, a method for removing fluorescence from the aqueous environment containing functionalized, encapsulated fluorescent nanocrystals comprises contacting the functionalized, encapsulated fluorescent nanocrystals with an effective amount of a disrupting agent (xe2x80x9clipolytic agentxe2x80x9d) to disrupt the liposome portion of the functionalized, encapsulated fluorescent nanocrystals thereby releasing the fluorescent nanocrystals into the aqueous environment with resultant precipitation out of solution.
As general guidance for producing functionalized, encapsulated fluorescent nanocrystals according to the present invention, there are various methods for forming liposomes which may be suitable for encapsulating fluorescent nanocrystals. In one embodiment, the lipids (e.g., phospholipids and sterol) for forming the liposomes, and the fluorescent nanocrystals to be encapsulated, are dissolved in a suitable solvent (e.g., chloroform), and the solvent is evaporated in vacuo to result in a film comprising the lipids and fluorescent nanocrystals (xe2x80x9cdried lipid mixture filmxe2x80x9d). For example, the lipids, fluorescent nanocrystals, and the organic solvent may be added to and mixed in a rotoevaporator flask, and dried under vacuum in a rotary evaporator until the contents form a thin homogenous film. Alternatively, a dried lipid mixture film may be formed by forming a dried lipid film, and adding to that dried lipid film a dried preparation of fluorescent nanocrystals. An aqueous solution is then added to the dried lipid mixture film, and the film is allowed to hydrate (e.g., for between 10-30 minutes at room temperature) resulting in spontaneous formation of liposomes which encapsulate fluorescent nanocrystals. The lipid dispersion may then be vigorously vortexed (e.g., 45 to 60 minutes) to facilitate continued formation of functionalized, encapsulated fluorescent nanocrystals. If desired, the lipid bilayers may be annealed by heating the dispersion to about 45 to 50xc2x0 C. followed by a gradual cooling to about 4xc2x0 C. It will be apparent to one skilled in the art that one or more components for substitution of the liposome may be suspended in the aqueous solution prior to the addition of the aqueous solution to the dried lipid mixture film. Alternatively, the liposome portion of the functionalized, encapsulated fluorescent nanocrystals may be post-treated (treated subsequent to formation) with one or more components for substitution of the liposome portion. For example, an aqueous solution containing the one or more components for substitution may be in prolonged contact (e.g., incubated overnight) with the functionalized, encapsulated fluorescent nanocrystals. As another alternative embodiment, the aqueous solution containing the one or more components for substitution, and dried lipid mixture film may be mixed together and strongly vortexed, followed by extrusion of the mixture under pressure through a membrane filter (e.g., polycarbonate) of a desired pore size to obtain a solution containing functionalized, encapsulated fluorescent nanocrystals. In another alternative embodiment, the one or more components for substitution are suspended in an aqueous solution, and then lyophilized. The lyophilized residue is then dissolved in a glycerol buffer (e.g., a 2% glycerol solution containing 0.5 mM EDTA, pH 6.0), and filtered through a membrane filter (e.g., polycarbonate) of a desired pore size. The resultant filtrate is added to the dried lipid mixture film, and the resultant mixture is then hydrated with an aqueous solution and vortexed to form functionalized, encapsulated fluorescent nanocrystals. If desired, the formed functionalized, encapsulated fluorescent nanocrystals may then be extruded through a membrane filter (e.g., polycarbonate) of a desired pore size. In any of these embodiments, the functionalized, encapsulated fluorescent nanocrystals would remain soluble in the aqueous solution in which they are formed, whereas unencapsulated fluorescent nanocrystals may eventually precipitate; hence, a purification may be achieved between functionalized, encapsulated fluorescent nanocrystals and unencapsulated fluorescent nanocrystals.
As will be apparent to one skilled in the art, there are various known methods for producing liposomes that may also be useful for producing functionalized, encapsulated fluorescent nanocrystals. Such methods may include, but are not limited to, a vortexing method, an ultrasonic method, an extrusion method, a reverse-phase evaporation method, a solvent injection method, a surfactant (e.g., detergent)-removal method, an annealing method, and a forced extrusion following freeze-thaw cycles. Each method may offer an advantage; thus, a combination of methods may be desirable (for a review, see, Szoka and Papahadjopoulos, 1981, Chapter 3 in xe2x80x9cLiposomes: From Physical Structure to Therapeutic Applicationsxe2x80x9d, the contents of which are herein incorporated by reference). For example, sonication is a method used to produce liposomes of a relatively small size as compared to other methods; however, the size is largely heterogenous. By extrusion through a membrane of a defined size, or series of membranes with pores of decreasing diameter, size heterogeneity can be reduced, thereby resulting in liposomes of a well-defined and narrow size dispersion. In another example, sonication may result in liposomes incorporating structural defects. However, annealing (at a temperature above the Tc of the highest melting lipid in the mixture used to form the liposome; e.g., for 30 minutes) can stabilize liposomes (note though, annealing is generally not effective when the liposome is composed of an equimolar ratio of phospholipid and cholesterol). The above principles can be applied to methods for producing functionalized, encapsulated fluorescent nanocrystals.
The selection and molar ratio of the combination of lipids, with or without one or more components for substitution, for encapsulating fluorescent nanocrystals may depend on factors which include, but are not limited to, the application in which the functionalized, encapsulated fluorescent nanocrystals are to be used, the desired surface groups comprising one or more reactive functionalities, desired size and/or stability and/or surface charge of the functionalized, encapsulated fluorescent nanocrystals, and the one or more methods used to make the functionalized, encapsulated fluorescent nanocrystals. Although various combinations may be used, a preferred combination may be selected from two general groupings of suitable lipid mixtures for forming the functionalized, encapsulated fluorescent nanocrystals according to the present invention: a combination of phospholipids with a sterol, wherein a phospholipid in the greatest amount of the combination (as compared to the amounts of the one or more remaining phospholipids of the combination) is in approximate equimolar ratio with the sterol; and a combination of phospholipids with a sterol, wherein the sterol is not in approximate equimolar ratio with the phospholipid comprising the highest amount (concentration) in the combination (as compared to the amounts of the one or more remaining phospholipids of the combination). Either combination may further comprise one or more components for substitution of the liposome portion of the functionalized, encapsulated fluorescent nanocrystals, as described herein in more detail. For purposes of illustration only, and not limitation, combinations of lipids (including with exemplary molar ratios) that may be useful in making the compositions according to the present invention include, but are not limited to, phosphatidylcholine (xe2x80x9cPCxe2x80x9d)/cholesterol (xe2x80x9cchxe2x80x9d)/phosphatidylserine (xe2x80x9cPSxe2x80x9d), 5:4:1; PC/ch/phosphatidylglycerol (xe2x80x9cPGxe2x80x9d), 8:2:1.2 or 9:8:1 or 9:5:1; PC/ch/phosphatidylethanolamine, 6:2:2 or 5:4:1; and dipalmitoylPC/ch/phosphatidic acid, 7:2:1. In a preferred embodiment, a combination of lipids may further comprise one or more components for substitution which is added to the lipids in parts by weight (as expressed in relation to the lipid mixture wherein the total lipid mixture comprises 1 part by weight) in a range of from about 0.0001 to about 0.5, depending on the nature of the one or more components, and the intended function. A preferred combination of lipids and one or more components for substitution may be used to the exclusion of combinations other than the preferred combination in producing the functionalized, encapsulated fluorescent nanocrystals according to the present invention. Similarly, a preferred fluorescent nanocrystal may be used to the exclusion of a fluorescent nanocrystal other than the preferred fluorescent nanocrystal in producing the functionalized, encapsulated fluorescent nanocrystals according to the present invention.
In another preferred embodiment, the one or more affinity molecules desired to be incorporated as part of a functionalized, encapsulated fluorescent nanocrystals is added in the process of producing functionalized, encapsulated fluorescent nanocrystals. In this preferred embodiment, it is desirable that the affinity molecule be comprised of a hydrophobic portion and a hydrophilic portion so that its hydrophobic portion will facilitate interaction with the hydrophobic portion of the lipids in the lipid mixture in forming the liposome portion of the functionalized, encapsulated fluorescent nanocrystals; and its hydrophilic portion will extend out from the surface of the functionalized, encapsulated fluorescent nanocrystals. For example, a dried lipid mixture comprising dried lipids, a dried preparation of the fluorescent nanocrystals, and a dried (e.g., lyophilized) preparation of the affinity molecule comprising a protein (e.g., monoclonal antibody, or peptide, or glycoprotein, or lipoprotein, etc.) is hydrated by the addition of an aqueous solution (alternatively, the affinity molecule is suspended in the aqueous solution); and the resultant dispersion is then vigorously vortexed to facilitate formation of functionalized, encapsulated fluorescent nanocrystals which have incorporated in the liposome portion the one or more affinity molecules. It will be apparent to one skilled in the art that, as compared to neutral phospholipids (e.g., PC), anionic phospholipids (e.g., PG and PS) enhance the binding of the affinity molecule in the liposome portion of the functionalized, encapsulated fluorescent nanocrystals. It will be apparent to one skilled in the art that the amount of affinity molecule to be incorporated, and the content (ratio) and composition of the lipid mixture will depend on the specific affinity molecule to be incorporated as well as the desired application of use for the functionalized, encapsulated fluorescent nanocrystals. In an illustrative, non-limiting example, the lipid mixture may comprise PC/ch/PG (17:5:2.5) and the affinity molecule comprises a peptide in an amount that is in a range of from about 0.001 mg/ml to about 1 mg/ml. As described in more detail herein, one or more components for substitution may be added. Additionally, if desired, the functionalized, encapsulated fluorescent nanocrystals may be subjected to a purification process such as size exclusion, or separation by function, or other method known in the art for purification.
The following examples are provided to further describe the invention, but are not to be considered limitative of the invention.